Conjugation of biomolecules using Diels-Alder cycloaddition

ABSTRACT

A method is provided for covalently linking carbohydrates, proteins, nucleic acids, and other biomolecules under neutral conditions, using a Diels-Alder cycloaddition reaction. In an example, activated carbon-carbon double bonds were attached to free amino sites of a carrier protein, and a conjugated diene was attached to a carbohydrate hapten. Spontaneous coupling of the carbohydrate and the protein components under very mild conditions provided glycoconjugates containing up to 37 carbohydrate hapten units per carrier protein molecule. The method is also applicable to the immobilization of biomolecules on gel or solid supports. The conjugated products are useful as immunogens and as analytical and diagnostic reagents.

FIELD OF THE INVENTION

[0001] The invention is in the field of bioorganic chemistry, morespecifically the field of conjugation of biomolecules. The conjugatedproducts prepared by the methods of the invention are useful, forexample, as inoculants for the generation of antibodies, and asvaccines. The methods of the invention may also be used to immobilizebiomolecules on solid supports. The immobilized biomolecules are usefulin many fields, such as for example catalysis, separation, analysis, anddiagnostics.

BACKGROUND

[0002] The conjugation of biomolecules to solid and gel supports is acommon operation in many laboratories, and many methods have beendeveloped for this purpose. Immobilization of enzymes (I. Chernukhin, E.Klenova, Anal. Biochem. 2000, 280:178-81), oligonucleotides (J.Andreadis; L. Chrisey, Nucleic Acids Res. 2000, 28:e5; A. Drobyshev etal., Nucl. Acids. Res. 1999, 27:4100-4105), antibodies (P. Soltys, M.Etzel, Biomaterials 2000, 21:37-48), and antigens (M. Oshima, M. Atassi,Immunol. Invest. 1989, 18:841-851) on solid and gel supports enables thepreparation of useful products such as chromatographic media (Meth.Enzym., W. Jakoby, M. Wilchek, eds., 1974, 34, Academic Press, NY),catalysts (T. Krogh et al., Anal. Biochem., 1999, 274:153-62),biosensors (J. Spiker, K. Kang, Biotechnol. Bioeng. 1999, 66:158-63),and numerous diagnostic (G. Ramsay, Nature Biotechnol., 1998, 16:40-44)and research tools (C. Bieri et al., Nature Biotechnol. 1999,17:1105-1108). Even whole cells may be immobilized by such methods (E.Olivares, W. Malaisse, Int. J. Mol. Med. 2000, 5:289-290).

[0003] The most robust form of attachment of a biomolecule to a surfaceor other support is via covalent bonds. Typically, such bonds areheteroatom-based (e.g., amide, ester, and disulfide bonds), because suchbonds are easily formed under mild conditions. Non-covalent attachmentvia specific binding pairs (e.g., biotin-avidin or antibody-antigeninteractions) is also commonly employed, but such methods still requireinitial conjugation of the specific binding pairs to the biomolecule andsupport. The use of carbon-carbon bonds for this purpose is very rare,because formation of C—C bonds is more difficult, especially under themild aqueous conditions appropriate for working with proteins.

[0004] The use of the Diels-Alder reaction to attach a member of aspecific binding pair has been described. In this report (M. N. Yousafand M. Mrksich, J. Am. Chem. Soc., 1999, 121:4286), a Diels-Alderreaction was used to covalently attach a biotinylated diene to animmobilized dienophile, and the immobilized biotin was subsequently usedto non-covalently immobilize streptavidin. These workers have morerecently used a Diels-Alder reaction to immobilize the peptide RGDS on aself-assembled alkanethiol monolayer on a gold surface (M. N. Yousaf, B.T. Houseman, M. Mrksich, Angew. Chem. Int. Ed. Engl., 2001, 40:1093).The use of the Diels-Alder reaction to effect the actual covalentcoupling or immobilization event of large biomolecules, however, had notpreviously been described.

[0005] The conjugation of biomolecules to one another is likewise a verycommon procedure, and is subject to most of the concerns and limitationsdescribed above for biomolecule immobilization. Covalent attachment ofhaptens to proteins has been a target of synthetic endeavors since thediscovery by Landsteiner that this process can convert non-immunogenicmolecules to immunogenic materials (K. Landsteiner, H. Lampl, Biochem.Zeitschr. 1918, 86:343). The application of this concept tocarbohydrates by Goebel and Avery revealed that covalentcarbohydrate-protein conjugates are immunogenic and can generateanti-carbohydrate antibodies (W. Goebel, J. Exp. Med. 1940, 72:33). Theuse of Landsteiner's principle has led to the development ofcarbohydrate-protein conjugates that are valuable tools in glycomedicalresearch, and that are useful as pharmaceuticals. In particular, proteinconjugates of fragments of the capsular polysaccharide of Haemophilusinfluenzae type b have become established as successful vaccines (J.Robbins et al., J. Am. Med. Assoc. 1996, 276:1181). Several otherbacterial saccharide-protein conjugates are in various stages ofclinical studies (E. Konadu et al., J. Infect. Dis. 1998, 177:383-387;E. Konadu et al., Infect. Immun. 2000, 68:1529-1534) while numerousothers are in the pre-clinical phase (V. Pozsgay et al., Proc. Natl.Acad. Sci. USA 1999, 96:5194).

[0006] The choice of methods for covalent bond formation betweenbiomolecules such as carbohydrates and proteins is restricted by theirlimited solubility in organic solvents, and in many cases by their pHand temperature sensitivity. In almost all cases, water is the onlysolvent that can be used for conjugation of carbohydrates or proteins,and the conditions are usually limited to temperatures under 50° C. andpH values between 6 and 8.

[0007] Numerous methods have been developed for the attachment ofpolysaccharides to proteins (C. Peeters et al., in Vaccine Protocols, A.Robinson et al, Eds., 1996Humana Press, NJ, p. 111; W. Dick, Jr., M.Beurret, in Contrib. Microbiol. Immunol., J. Cruse and R. Lewis, eds.,1989, 10:48-114, Karger, Basel; H. Jennings, R. Sood, inNeoglycoconjugates. Preparation and Applications, Y. Lee, R. Lee, eds.,Academic Press, New York, 1994, p. 325). However, only a few of thesemethods are capable of coupling oligosaccharides to carriers in asite-selective fashion. Most prominent among these is reductiveamination, which converts the reducing-end residue of the polysaccharideinto a polyhydroxy alkylamino moiety, which unfortunately causes theloss of this unit as a true carbohydrate in the resulting glycoconjugate(V. Pozsgay, Glycoconjugate J. 1993, 10:133).

[0008] This problem can be solved by chemical synthesis ofoligosaccharide glycosides with aglycons that bear a (latent) reactivegroup. Examples include alkenyl groups (M. Nashed, Carbohydr. Res. 1983,123:241-246; J. Allen, S. Danishefsky, J. Am. Chem. Soc. 1999,121:10875), 3-aminopropyl (G. Veeneman et al., Tetrahedron 1989,45:7433), 4-aminophenylethyl (R. Eby, Carbohydr. Res. 1979, 70:75),4-aminophenyl (S. Stirm et al., Justus Liebigs Ann. Chem. 1966,696:180), 6-aminohexyl (J. Hermans et al., Rec. Trav. Chim. Pays-Bas1987, 106:498; R. Lee et al., Biochemistry 1989, 28:1856),5-methoxycarbonylpentyl (S. Sabesan, J. Paulson, J. Am. Chem. Soc. 1986,108:2068; V. Pozsgay, Org. Chem. 1998, 63:5983), 8-methoxycarbonyloctyl(R. Lemieux et al., J. Am. Chem. Soc. 1975, 97, 4076; B. Pinto et al.,Carbohydr. Res. 1991, 210, 199) 4-aminobenzyl (W. Goebel, J. Exp. Med.1940, 72:33), ω-aldehydoalkyl (V. Pozsgay, Glycoconjugate J. 1993,10:133), 3-(2-aminoethylthio)propyl (Y. Lee, R. Lee, Carbohydr. Res.1974, 37:193), 2-chloroethylthioglycosides (M. Ticha et al.,Glycoconjugate J. 1996, 13:681) and 1-O-succinimide derivatives (M.Andersson, S. Oscarson, Bioconjugate Chem. 1993, 4:246; B. Davis, J.Chem. Soc. Perkin I 1999, 3215).

[0009] These aglycons introduce spacers that can be linked to a proteineither directly or after insertion of a secondary linker. For thispurpose the use of an activated dicarboxylic acid has been reported (R.van den Berg et al., Eur. J. Org. Chem. 1999, 2593-2600). In anotherprocedure, a sulfhydryl group at the terminal position of the spacerallows the formation of a disulfide bridge with proteins using thedithiopyridyl method (J. Evenberg et al., J. Infect. Dis. 1992, 165(sup.1):S152). In a related protocol, a thiolated protein is coupled with amaleimido-derivatized saccharide (J. Mahoney, R. Schnaar, MethodsEnzymol. 1994, 242:17). N-acryloylamidophenyl glycosides may be coupledto unmodified proteins using a Michael addition (A. Romanowska et al.,Methods Enzymol. 1994, 242:90). As an alternative to glycosideformation, direct coupling of a carbohydrate to a linker via amide bondshas also been used (A. Fattom et al., Infect. Immun. 1992, 60:584-589),but this approach is limited to carboxyl-containing carbohydrates.

[0010] The yields of any of these methods rarely exceed 40%, and aregenerally in the 10-20% range (R. van den Berg et al., Eur. J. Org.Chem. 1999, 2593-2600), especially when medium or high carbohydrateloading in the conjugate is attempted. This problem is compounded by thefact that the oligosaccharide haptens, usually obtained in multistepsyntheses or by controlled degradation of polysaccharides, can rarely berecovered in their active or activable form after the couplingprocedure. An additional problem with most chemical coupling methodsemployed to date is the formation of cross-linked byproducts, due to thepresence of multiple reactive functional groups (e.g., amines, acids,hydroxyls, and sulfhydryls) on most biomolecules. Avoidance of thisproblem requires that the reactive groups be blocked, which requiresadditional processing steps and may alter the physicochemical andimmunological properties of the biomolecule. Thus, there remains a needfor a mild and site-selective method for coupling biomolecules to oneanother, which avoids the problems of low yields, crosslinking, and lossof starting materials. For similar reasons there remains a need for mildand selective methods for attaching biomolecules to surfaces and solidand gel supports.

BRIEF DESCRIPTION OF THE INVENTION

[0011] The present invention provides an experimentally simple protocolfor the covalent attachment of biomolecules to one another and tosupports, that can avoid many of the above-mentioned problems. Theinvention makes use of the well-known Diels-Alder cycloaddition reactionthat takes place between a double bond and a conjugated diene. Thisreaction has traditionally been carried out in organic solvents, but canproceed in aqueous solutions as well (R. Breslow, D. Rideout, J. Am.Chem. Soc. 1980, 102:7816; A. Lubineau, J. Auge, Top. Curr. Chem. 1999,206:1; P. Garner, in Organic Synthesis in Water, P. Grieco, ed., BlackieAcademic and Professional, London, 1998, p. 1.)

[0012] Carbohydrates have been employed as chiral auxiliaries and/orwater solubilizing agents for Diels-Alder reactions, wherein aconjugated diene system is converted to a glycoside prior to thecycloaddition (A. Lubineau et al., J. Chem. Soc. Perkin 1 1997,2863-2867; see also S. Pellegrinet, R. Spanevello, Org. Lett. 2000,2:1073-1076). As noted above, the Diels-Alder reaction has also beenused to covalently attach biotin to a support (M. N. Yousaf and M.Mrksich, J. Am. Chem. Soc., 1999, 121:4286). However, the Diels-Alderreaction has not previously been extended to the direct covalentconjugation of biopolymers or other types of polymeric materials. Amongthe advantages of the method of the invention are the mild and neutralconditions, good yields, negligible cross-linking, and facile recoveryof excess and/or unreacted biomolecules in their conjugatable form.

[0013] The invention also provides conjugated biomolecules, which areuseful as immunostimulatory agents for production of antibodies andinduction of immunity, methods of inducing antibody production with theconjugated biomolecules, and vaccine compositions comprising theconjugated biomolecules.

[0014] The invention also provides polyclonal and monoclonal antibodiesgenerated by administration of the conjugated biomolecules to a mammal,and methods of using the induced antibodies for inducing passiveimmunity. The antibodies are useful of therapeutic, diagnostic, andanalytical purposes.

[0015] The invention also provides immobilized biomolecules, and methodsfor their preparation, which are useful in many areas, such aschromatographic media, catalysts, components of diagnostic devices,biosensors, and as research tools.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The invention provides a new method for conjugation ofbiomolecules based on the Diels-Alder cycloaddition reaction. Thetechnique involves the introduction of an activated double bond into afirst biomolecule component, and a conjugated diene into a secondbiomolecule component, which are to be covalently linked together. Thediene- and dienophile-modified biomolecules may then be purified to theextent desired. The two components are then simply combined underneutral conditions, and the cycloaddition reaction is allowed toproceed.

[0017] As used herein, the term “biomolecule” refers generally to thelarge, complex molecules produced within living cells, and to syntheticand semi-synthetic analogues thereof. Examples include proteins,peptides, oligo- and poly-saccharides, and oligo- and poly-nucleicacids, and various combinations thereof such as for exampleglycoproteins and nucleoproteins. Larger complexes, such as ribosomes,cellular substructures, and even entire cells are intended to fallwithin the meaning of the term as well.

[0018] In certain of the examples presented, the diene moiety isintroduced into a carbohydrate component and the activated double bondinto a polypeptide component. This may be reversed if desired, as amatter of convenience or if required by the synthetic design, as shownin another of the examples. The cycloaddition step proceeds in mostcases under neutral conditions, at or below physiological temperatures.Where they are of sufficiently low molecular weight, unreactedcomponents can be recovered for re-use by simple diafiltration of thecoupling reaction mixture.

[0019] The embodiment of the invention disclosed in certain of theexamples below incorporates an electron-deficient carbon-carbon doublebound into a protein, with human serum albumin (HSA) being used as anexample and the commercially available reagent 3-sulfosuccinimidyl4-maleimidobutyrate being used as the reagent. In these embodiments, thediene component of the Diels-Alder reaction is incorporated into acarbohydrate, with derivatives of trans,trans-hexa-2,4-dien-1-ol,1-amino-hexa-2,4-diene, and octa-2,4-dienoic acid hydrazide being usedas dienes. It will be understood that in general, the Diels-Alderreaction will occur between sterically accessible dienes and dienophilesregardless of the nature of the attached biomolecules, and that byappropriate selection of reagents both homoconjugates andheteroconjugates of proteins, carbohydrates, and oligonucleotides can becarried out by the methods of this invention.

[0020] The methods of the invention unexpectedly provide for a degree ofcontrol over the rate of coupling of the diene and dienophilecomponents, simply by modification of the linker moieties. The molecularweight of the conjugate approaches a limiting value as the couplingreaction proceeds, which depends on the mass of the carbohydrate beingattached. The observed increase in molecular weight as a function oftime may therefore be fit to a pseudo-first order reaction kineticequation of the form

Δmw=Δmw _(max)(1−e ^(−kt))

[0021] where Δmw is the increase in molecular weight, Δmw_(max) is themaximum increase that could be obtained if all the protein-linkedstarting material were to react, k is the rate constant, and t is time.The curves drawn through the data of FIGS. 1 and 2 (see Example 5 forthe experimental details) were obtained by fitting the parameters ofthis equation to the data. The results of this fitting are summarized inTable 2.

[0022] The faster incorporation of construct 27 relative to 8 is aninteresting observation. The observed difference apparently correlateswith the distance between the hydrophilic rhamnose moiety and thehydrophobic diene part of the molecule. According to Breslow, theaccelerating effect of water on the rate of Diels-Alder reactions is dueto hydrophobic packing of the reactants (R. Breslow, D. Rideout, J. Am.Chem. Soc. 1980, 102:7816). Such an effect should be more pronounced incompound 27, where the diene sector is more isolated from thehydrophilic portion, than in compound 8.

[0023] The Diels-Alder reaction requires a highly organized transitionstate, and the ability of the components of the present invention toachieve the proper geometry for cycloaddition despite the mass and bulkof the attached biomolecules is remarkable.

[0024] In a preferred embodiment of the invention, one of thebiomolecules to be linked is a hapten or antigen, and the other is acarrier. In a particularly preferred embodiment, the hapten or antigenis a polysaccharide moiety. Examples of antigenic polysaccharides arethe capsular polysaccharides of Haemophilus influenzae type b, Neisseriameningitidis, Group B Streptococci, Salmonella typhi, E. coli, andPneumococci.

[0025] Carriers are chosen to increase the immunogenicity of the haptenor antigen, and/or to raise antibodies against the carrier itself whichmay be medically beneficial. Carriers that fulfill these criteria areknown in the art (see, e.g. A. Fattom et al., Infect. Immun. 1990, 58,2309-2312; Devi, J. Robbins, R. Schneerson, Proc. Natl. Acad. Sci. USA1991, 88:7175-7179; S. Szu, X et al., Infect. Immun., 1991,59:4555-4561; S. Szu et al., J. Exp. Med., 1987, 166:1510-1524). Acarrier can be a natural, semi-synthetic, or synthetic materialcontaining one or more functional groups, for example primary and/orsecondary amino groups, azido groups, hydroxyl groups, or carboxylgroups, to which a diene or dienophile Diels-Alder reactant moiety canbe attached. The carrier can be water soluble or insoluble, and ispreferably a polypeptide.

[0026] Examples of water soluble polypeptide carriers include, but arenot limited to, natural, synthetic, or semisynthetic peptides orproteins from bacteria or viruses, e.g., bacterial, bacterial outermembrane proteins, bacterial toxins and toxoids such as tetanustoxin/toxoid, diphtheria toxin/toxoid, Pseudomonas aeruginosaexotoxin/toxoid/protein, pertussis toxin/toxoid, and Clostridiumperfringens exotoxins/toxoid. Viral proteins such as hepatitis B surfaceantigen and core antigen may also be used as carriers, as well asproteins from higher organisms such as keyhole limpet hemocyanin,horseshoe crab hemocyanin, edestin, mammalian serum albumins, mammaliangamma-globulins, and IgG.

[0027] Polysaccharide carriers include, but are not limited to, dextran,capsular polysaccharides from microorganisms such as the Vi capsularpolysaccharide from S. typhi, which is described in U.S. Pat. No.5,204,098, (incorporated by reference herein); Pneumococcus group 12(12F and 12A) polysaccharides; Haemophilus influenzae type dpolysaccharide; and certain plant, fruit, or synthetic oligo- orpolysaccharides which are immunologically similar to capsularpolysaccharides, such as pectin, D-galacturonan, oligogalacturonate, orpolygalacturonate, for example as described in U.S. Pat. No. 5,738,855(incorporated by reference herein).

[0028] Examples of water insoluble carriers include, but are not limitedto, aminoalkyl agarose, e.g., aminopropyl or aminohexyl SEPHAROSE(Pharmacia Inc., Piscataway, N.J.), aminopropyl glass, cross-linkeddextran, and the like, to which a diene or dienophile can be attached.Other carriers may be used provided that a functional group is availablefor covalently attaching a diene or dienophile.

[0029] Examples of dienophiles include, but are not limited to,maleimides, acrylamides, azodicarboxylates, quinones, and1,2,4-triazoline-3,5-diones. Examples of dienes include, but are notlimited to, esters and glycosides of hexa-2,4-dien-1-ol,penta-2,4-dien-1-ol, furan-2-methanol, and furan-1-methanol; esters,amides, and hydrazides of octa-2,4-dienoic acid; and amides of1-aminohexa-2,4-diene and 1- and 2-aminomethylfuran. The above-mentionedamines may also be coupled with aldehydo-biomolecules via reductiveamination, and hydrazides may be attached to such biomolecules viacondensation.

[0030] The invention also provides biomolecule conjugates of generalformulas I and II below:

[0031] where R and R′ are independently H or methyl, or togetherconstitute CH₂, CH₂CH₂, SO₂, or O; X is CH or N; Y is N, CH═C, or NH—N;and B₁ and B₂ comprise biomolecules independently selected from thegroup consisting of polypeptides, carbohydrates, polysaccharides, andnucleic acids, and are optionally attached via a linker.

[0032] The invention also provides immobilized biomolecules of formulasI and II above, wherein one of B₁ and B₂ may be a solid or gel support.Examples of solid supports include, but are not limited to,aminopropylsilylated glass and silica surfaces, gold surfacesfunctionalized with thiol-bound linkers, functionalized macroporouspolystyrene beads, and surface-derivatized microtiter plate wells.Examples of gel supports include, but are not limited to, functionalizedagarose gels such as cyanogen bromide activated agarose, aminoethylagarose, and carboxymethyl agarose.

[0033] The formulas above are intended to indicate that the group B₁ maybe attached alpha or beta to the group R′ as shown below:

[0034] There are many known methods of attachment of small molecules tobiomolecules, there are many known linker moieties for attachment ofchemical moieties to biomolecules, and there are many known dienes anddienophiles that readily take part in cycloaddition reactions at or nearroom temperature. Those skilled in the art will thus appreciate thatthere are many obvious combinations of attachment methods, linkers, anddiene and dienophile partners that may be employed in the method ofbiomolecule coupling disclosed herein, which are equivalent to theexamples provided. Such modifications of the disclosed methods andresulting compositions are intended to be within the scope and spirit ofthe present invention.

[0035] It is another object of the invention to provide methods of usingthe polysaccharide-carrier conjugates of this invention for eliciting animmunogenic response in mammals, including but not limited to responseswhich provide protection against, or reduce the severity of, bacterialand viral infections. The pharmaceutical compositions of this inventionare expected to be capable, upon injection into a mammal, of inducingserum antibodies against the polysaccharide component of the conjugate.

[0036] The invention also provides methods of using such conjugates,and/or pharmaceutical compositions comprising such conjugates, to inducein mammals, in particular, humans, the production of antibodies whichimmunoreact with the polysaccharide component of the conjugates.Antibodies which immunoreact with a bacterial or viral polysaccharideare useful for the identification or detection of microorganismsexpressing the polysaccharide, and/or for diagnosis of infection.Antibodies against the polysaccharide may be useful in increasingresistance to, preventing, ameliorating, and/or treating illnessescaused by microorganisms or viruses that express the polysaccharide.

[0037] The compositions of this invention are intended for activeimmunization for prevention of infection, and for preparation of immuneantibodies. The compositions of this invention are designed to induceantibodies specific to microorganisms expressing the polysaccharidecomponent of the conjugate, and to confer specific immunity againstinfection with such microorganisms.

[0038] This invention also provides compositions, including but notlimited to, mammalian serum, plasma, and immunoglobulin fractions, whichcontain antibodies which are immunoreactive with the polysaccharidecomponent of the conjugates of this invention, and which preferably alsocontain antibodies which are immunoreactive with the protein component.These antibodies and antibody compositions may be useful to prevent,treat, or ameliorate infection and disease caused by the microorganism.The invention also provides such antibodies in isolated form. Theinvention further provides methods of inducing in mammals antibodieswhich immunoreact with a polysaccharide, the methods comprisingadministering to a mammal a composition of the invention.

[0039] The invention also provides monoclonal antibodies, preferablyproduced by hybridomas, which immunoreact with a polysaccharide. Thenucleic acid sequences encoding these antibodies are obtained from amammal in which the production of anti-polysaccharide antibodies hasbeen induced by administering a composition of the invention.

[0040] As used herein, the terms “immunoreact” and “immunoreactivity”refer to specific binding between an antigen or antigenicdeterminant-containing molecule and a molecule having an antibodycombining site, such as a whole antibody molecule or a portion thereof.

[0041] As used herein, the term “antibody” refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules. Exemplary antibody molecules are intact immunoglobulinmolecules, substantially intact immunoglobulin molecules and portions ofan immunoglobulin molecule, including those portions known in the art asFab, Fab′, F(ab′)₂ and F(v), as well as chimeric antibody molecules.

[0042] Polymeric Carriers

[0043] Carriers are chosen to increase the immunogenicity of thepolysaccharide and/or to raise antibodies against the carrier which aremedically beneficial. Carriers that fulfill these criteria arewell-known in the art. A polymeric carrier can be a natural or asynthetic material containing one or more functional groups, for exampleprimary and/or secondary amino groups, azido groups, or carboxyl groups,to which a diene or dienophile component can be attached. Carriers canbe water soluble or insoluble. The examples below employ proteins ascarriers.

[0044] Regardless of the precise method used to prepare the conjugate,after the Diels-Alder coupling reaction has been carried out theunreacted materials are preferably removed by routine physicochemicalmethods, such as for example dialysis, gel filtration or ion exchangecolumn chromatography, depending on the materials to be separated. Thefinal conjugate consists of the polysaccharide and the carrier boundthrough a Diels-Alder adduct.

[0045] Dosage for Vaccination

[0046] The present inoculum contains an effective, immunogenic amount ofa polysaccharide-carrier conjugate. The effective amount ofpolysaccharide-carrier conjugate per unit dose sufficient to induce animmune response depends, among other things, on the immunogenicity ofthe polysaccharide, the species of mammal inoculated, the body weight ofthe mammal, and the chosen inoculation regimen, as is well known in theart. Inocula typically contain polysaccharide-carrier conjugates withconcentrations of polysaccharide from about 1 micrograms to about 500micrograms per inoculation (dose), preferably about 3 micrograms toabout 50 micrograms per dose, and most preferably about 5 micrograms to25 micrograms per dose.

[0047] The term “unit dose” as it pertains to the inocula refers tophysically discrete units suitable as unitary dosages for mammals, eachunit containing a predetermined quantity of active material(polysaccharide) calculated to produce the desired immunogenic effect inassociation with the required diluent.

[0048] Inocula are typically prepared in physiologically and/orpharmaceutically tolerable (acceptable) carriers, and are preferablyprepared as solutions in physiologically and/or pharmaceuticallyacceptable diluents such as water, saline, phosphate-buffered saline, orthe like, to form an aqueous pharmaceutical composition. Adjuvants, suchas aluminum hydroxide, QS-21, TiterMax™ (CytRx Corp., Norcross Ga.),Freund's complete adjuvant, Freund's incomplete adjuvant, interleukin-2,thymosin, and the like, may also be included in the compositions.

[0049] The route of inoculation may be by intramuscular or subcutaneousinjection or the like, so long as it results in eliciting antibodiesreactive against the polysaccharide component. It is anticipated that insome cases the composition can be administered orally or intranasally,for example when mucosal immunity is to be induced. In order to increasethe antibody level, a second or booster dose may be administeredapproximately 4 to 6 weeks after the initial administration. Subsequentdoses may be administered as deemed necessary by the practitioner.

[0050] Antibodies

[0051] An antibody of the present invention is typically produced byimmunizing a mammal with an immunogen or vaccine containing apolysaccharide-carrier conjugate, preferably a polysaccharide-proteinconjugate, to induce in the mammal antibody molecules havingimmunospecificity for the polysaccharide component of the conjugate.Antibody molecules having immunospecificity for the protein carrier mayalso be produced. The antibody molecules may be collected from themammal and, optionally, isolated and purified by methods known in theart.

[0052] Human or humanized monoclonal antibodies are preferred, includingbut not limited to those identified by phage display technology, andincluding but not limited to those made by hybridomas and by mice withhuman immune systems or human immunoglobin genes. The antibody moleculesof the present invention may be polyclonal or monoclonal. Monoclonalantibodies may be produced by methods well-known in the art. Portions ofimmunoglobulin molecules, such as Fabs, may also be produced by methodsknown in the art.

[0053] An antibody of the present invention may be contained in bloodplasma, serum, hybridoma supernatants and the like. Alternatively, theantibodies of the present invention are isolated to the extent desiredby well known techniques such as, for example, ion chromatography oraffinity chromatography. The antibodies may be purified so as to obtainspecific classes or subclasses of antibody such as IgM, IgG, IgA, IgG₁,IgG₂, IgG₃, IgG₄ and the like. Antibodies of the IgG class are preferredfor conferring passive immunity. The antibodies of the present inventionhave a number of diagnostic and therapeutic uses. The antibodies can beused as an in vitro diagnostic agents to test for the presence ofmicroorganisms in biological samples or in water or food samples, instandard immunoassay protocols. Such assays include, but are not limitedto, agglutination assays, radioimmunoassays, enzyme-linked immunosorbentassays, fluorescence assays, Western blots and the like. In one suchassay, for example, the sample is contacted with first antibodies of thepresent invention, and a labeled second antibody is used to detect thepresence of polysaccharides to which the first antibodies have bound.

[0054] Such assays may be, for example, of direct format (where thelabeled first antibody is reactive with the polysaccharide), an indirectformat (where a labeled second antibody is reactive with the firstantibody), a competitive format (such as the addition of a labeledpolysaccharide), or a sandwich format (where both labeled and unlabelledantibody are utilized), as well as other formats described in the art.

[0055] In providing the antibodies of the present invention to arecipient mammal, the dosage of administered antibodies will varydepending upon such factors as the mammal's age, weight, height, sex,general and specific medical conditions, and the like.

[0056] In general, it is desirable to provide the recipient with adosage of antibodies which is in the range of from about 1 mg/kg toabout 10 mg/kg body weight of the mammal, although a lower or higherdose may be administered. The antibodies of the present invention areintended to be provided to the recipient subject in an amount sufficientto prevent, or lessen or attenuate the severity, extent or duration ofthe infection.

[0057] In order to facilitate the administration of the conjugates ofthe invention to mammals, it is preferred that the conjugate beformulated with a pharmaceutically acceptable carrier. (Those skilled inthe art will appreciate that the term “carrier,” when used in thiscontext, has a different meaning than when it is used to refer to abiomolecule component of the conjugate.) Examples of pharmaceuticallyacceptable carriers include sterile water and saline, both of which maybe buffered with phosphate, citrate, and the like. The conjugates of theinvention may be provided in solution or suspension in apharmaceutically acceptable carrier, or they may be provided in dry formand reconstituted with the pharmaceutically acceptable carrier prior toadministration.

[0058] The administration of the conjugates and compositions of theinvention may be for prophylactic or therapeutic purposes. When providedprophylactically, the agents are provided in advance of any symptom. Theprophylactic administration of the agent serves to prevent or ameliorateany subsequent infection. When provided therapeutically, the agent isprovided at (or shortly after) the onset of a symptom of infection.

[0059] For all therapeutic, prophylactic and diagnostic uses, thepolysaccharide-carrier conjugates of this invention, as well asantibodies and other necessary reagents and appropriate devices andaccessories may be provided in kit form so as to be readily availableand easily used.

[0060] The examples below will be understood to be merely representativeof the invention, and are not intended to limit the scope of theappended claims in any way.

[0061] Dieneophile component 2:

[0062] Treatment of human serum albumin (HSA) with a 1.6 molar excess(based on 58 available amino groups) of 3-sulfosuccinimidyl4-maleimidobutyrate (1) in a pH 7.5 phosphate buffer afforded theintermediate 2, which contained an average of 38 maleimido moieties perprotein molecule, as indicated in the formula (determined by MALDI-TOFmass spectroscopy).

[0063] Diene component 8:

[0064] The phenylthio rhamnoside 3 was prepared as described previously(V. Pozsgay, Carbohydr. Res., 1992, 235:295). Rhamnoside 3 was treatedwith acetic anhydride and pyridine to afford 4, ¹H NMR (CDCl₃, δ)8.11-7.26 (m, 15H), 5.79 (dd, 1H), 5.64-5.52 (m, 3H), 5.53 (t, 1H,J=10.0 Hz), 4.69 (dq, 1H), 1.89 (s, 2H), 1.35 (d, 3H, J=6.3 Hz), ¹³C(CDCl₃, δ) 170.1, 165.7, 165.5, 133.5-127.9, 85.8, 72.1, 71.9, 69.5,68.1, 20.6, 17.6.

[0065] From 4 the hemiacetal 5 was obtained by hydrolysis with mercurictrifluoroacetate (L. Yan, D. Kahne, J. Am. Chem. Soc. 1996, 118:9239),¹H NMR (CDCl₃, δ) 8.1-7.4 (m, 10H), 5.71 (dd, 1H, J=3.4 Hz, J=9.9 Hz),5.78 (dd, 1H), 5.49 (t, 1H, J=9.9 Hz), 5.38 (br d, 1H), ¹³C (CDCl₃, δ)170.3, 165.8, 92.2, 71.9, 71.0, 68.9, 66.7, 20.7, 17.7.

[0066] Hemiacetal 5 was converted to the trichloroacetimidate 6, andglycosylation of trans,trans-hexa-2,4-dien-1-ol with 6 usingCF₃SO₃Si(CH₃)₃ as the activator afforded the glycoside 7. The acetylgroups were then removed by treatment with NaOMe to afford the dienerhamnoside 8. ¹H NMR (D₂O, δ) 6.33 (dd, 1H, J=9.5 Hz, J=10.2 Hz), 6.16(ddd, 1 H, J=1.6 Hz, J=10.2 Hz, J=14.8 Hz), 5.85 (m, 1H), 5.69 (m, 1H),4.83 (d, 1H, J=1.7 Hz,) 4.21 (dd, 1H, J=6.4 Hz, J=12.4 Hz), 4.07 (dd,1H, J=7.1 Hz, J=12.4 Hz), 3.91 (dd, 1H, J=1.7 Hz, J=3.4 Hz), 3.69 (dq,1H), 3.73 (dd, 1H, J=3.4 Hz, J=9.6 Hz), 3.44 (t, 1 H, J=9.6 Hz), 1.75(d, 3H, J=6.8 Hz), 1.28 (d, 3H, J=6.3 Hz), ¹³C (D₂O, δ) 136.7, 132.7,131.1, 125.6, 99.8, 72.8, 71.1, 71.0, 70.9, 69.4, 68.6, 18.2, 17.4.

[0067] Coupling reaction:

[0068] An excess of 8 was treated in an aqueous solution with themaleimido-derivatized protein 2. The average incorporations of thehapten, as a function of time and temperature, are shown in Table 1.This data was obtained from the average molecular mass of the conjugatesdetermined by the MALDI-TOF method. As expected for a concertedcycloaddition reaction, the incorporation level depends on the reactiontime and temperature. At room temperature, approximately 63% of theavailable dienophile moieties in the protein participated in adductformation within 36 h, while at 40° C. almost complete utilization ofthese moieties occurred after four days (Table 1). The unreacted diene 8was recovered by diafiltration, and the conjugate 9 (free amino groupsnot shown) was then obtained as a white solid after freeze-drying. TABLE1 Time and temperature dependence of the cycloaddition between 2 and 8Composition of the conjugate (mol hapten/mol albumin) Time (h) 22° C.40° C.  36 24 27 100 28 37

EXAMPLE 2

[0069] Dienophile Component:

[0070] The glycoside of 6-hydroxyhexanoic acid hydrazide with thetetramer of(α-L-rhamnopyranosyl)-(1→2)-(α-D-galactopyranosyl)-(1→3)-(α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoseis prepared, according to the procedure disclosed in internationalpatent application WO 99/03871. Treatment with maleic anhydride providesan N-terminal maleimide derivative 10.

[0071] Diene component:

[0072] Trans,trans-hexa-2,4-dien-1-ol and succinic anhydride are reactedin the presence of N,N-dimethylaminopyridine to providetrans,trans-hexa-2,4-dien-1-ol monosuccinate. An aqueous solution of anexcess of the monosuccinate is activated with1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and coupled with humanserum albumin, to provide a poly(diene) derivative 11.

[0073] Coupling Reaction:

[0074] An aqueous solution of 11 and excess 10 is incubated at 35° C.for 4 days, and the resulting conjugate 12 (n≦58) is purified bydiafiltration and lyophilized. The conjugate 12 is expected to be usefulfor inducing antibodies against Shigella dysenteriae.

EXAMPLE 3

[0075] Diene component:

[0076] The Vi capsular polysaccharide of Salmonella typhi (PasteurMerieux Serums et Vaccins, Lyon FR) is dissolved in water, excess2,4-hexadienylamine is added, and the solution buffered to pH 5.0. Aslight excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide inwater is added. After 4 h at 37°, the mixture is adjusted to pH 7.5 anddialyzed to remove excess reagents. The resulting solution of 13 is usedimmediately.

[0077] Dienophile Component:

[0078]Pseudomonas aeruginosa recombinant exoprotein A (Fattom et al.,Infect. Immun. 1992, 60:584-589) is treated with excess3-sulfosuccinimidyl 4-maleimidobutyrate (1) as in Example 1, followed bydialysis and lyophilization to provide a maleimide derivatized protein14.

[0079] Coupling Reaction:

[0080] An excess of the diene 14 is added to the solution of dienophilecomponent 13. After 4 days at 37° C., the mixture is concentrated, andconjugate 15 is purified by size exclusion chromatography on SEPHACRYLS-1000™(Pharmacia, Piscataway N.J.). The conjugate 15 is expected to beuseful for inducing antibodies against Salmonella typhi.

[0081] Diene Component:

[0082] To a solution of methyl octa-4,6-dienoate (16) (6 g) in methanol(10 ml) was added hydrazine (3 ml) at room temperature. After 24 h, thesolution was diluted with water (50 ml). The crystalline precipitate wasisolated by filtration to afford octa-4,6-dienoic acid hydrazide (17) ascolorless microcrystals, yield 5.7 g. Dextran (nominal MW 10 kDa,Pharmacia) was diafiltered through a YM10 membrane (MW cutoff 10 kDa,Millipore) using 3 changes of water. The solution that passed throughthe membrane was diafiltered through a YM3 (MW cutoff 3000 Da) membrane,using five changes of water. The material retained by the membrane waslyophilized. A stirred solution of the dextran 18 thus obtained (22.5mg, 2.25 μmol, corresponding to 139 μmol of glucose) in H₂O (2.2 ml) at5° C. (ice bath) was equipped with a temperature-sensing pH electrode,and 0.1 M NaOH was added with a 100 μL microsyringe until the pH reached10.5. To this solution was added BrCN (50 μL of a 5 M solution inacetonitrile). The pH of the solution was maintained between 10.5 and10.8 by addition of 0.1 M NaOH with a microsyringe. After 6 min, thesolution was adjusted to pH 8.5 by addition of pH 8.0 phosphate buffer(ca. 1 ml). To the reaction mixture was immediately added a solution ofocta-4,6-dienoic acid hydrazide (3.5 mg, 25 μmol) in 0.5 ml dimethylsulfoxide. The pH of the reaction mixture rose to 8.95, at a temperatureof 9° C. The ice bath was removed and the stirred solution was allowedto reach 25° C. After 2.5 h, the pH was 8.18 and remained unchanged over5 min. The solution of 20 was diafiltered through a YM-3 membrane using5 changes of water (4 ml each).

[0083] Dienophile Component:

[0084] A stirred solution of human serum albumin (11.4 mg, 0.17 μmol,9.88 μmol of amino groups) in pH 7.5 buffer (1 ml) was treated at 5° C.(ice-bath) with 3-sulfosuccinimidyl 4-maleimidobutyrate (1) (0.52 mg,1.36 μmol). The solution was stirred for 15 min at 5° C. then foranother 15 min at room temperature followed by diafiltration through aYM-10 membrane using 5 changes of water (4 ml each).

[0085] Coupling Reaction:

[0086] The residual solutions containing the modified dextran 20 and themodified human serum albumin were combined. The total volume of thecombined solution was approx. 1 ml. After 22 h at room temperature, thereaction mixture was diafiltered through a YM-10 membrane using 6changes of H₂O (5 ml each), and the residue was freeze-dried. MALDI massspectroscopy showed that most of the albumin is consumed. The conjugate21 that is formed has average molecular weight of 90 kDa.

[0087] Diene Component:

[0088] 6-Hydroxyhexanoic acid 23 (S. Sabesan, J. C. Paulson, J. Am.Chem. Soc., 1986, 108:2068) and glycosyl chloride 22 (V. Pozsgay,Glycoconj. J., 1993, 10:133), in methylene chloride at −40° C., weretreated with silver triflate and 2,6-di-t-butyl-4-methylpyridine for 10min. to afford glycoside 24. Deprotection with a catalytic amount ofNaOMe in MeOH at 23° C. for 24 hr, followed by treatment withethylenediamine in ethanol at 80° C. for 12 hr, provided 25 in 74%overall yield. ¹H NMR (500 MHz, CD₃OD): δ=4.64 (br s, 1H), 3.78 (dd,1H), 3.66 (m, 1H), 3.55 (dq, 1H), 3.39 (m, 1H), 3.35 (t, 1H), 3.36 (t,2H), 2.76 (t, 2H), 2.21 (t, 2H), 1.54-1.68 (m, 4H), 1.35-1.46 (m, 2H),1.24 (d, 3H).

[0089] Octadienoic acid 26 was prepared from the corresponding methylester (T. Hudlicky et al., J. Org. Chem., 1980, 45:5020). Acylation of25 with 26 (dicyclohexyl-carbodiimide, EtOAc, MeOH) afforded theglycoside diene 27 in 92% yield, ¹H NMR (500 MHz, CD₃OD): δ=6.09 (m,2H), 5.70 (m, 1H), 5.57 (m, 1H), 4.77 (br s, 1H), 3.92 (dd, 1 H),3.63-3.75 (m, 4H), 3.53 (m, 1H), 3.43 (t, 1H), 3.32 (m, 4H), 2.32 (m,4H), 2.22 (m, 2 H), 1.72 (d, 3H), 1.53-1.67 (m, 4H), 1.30-1.42 (m, 2H),1.29 (d, 3H).

[0090] Dienophile Component:

[0091] A stirred solution of human serum albumin in pH 7.5 buffer (1 ml)was treated at 5° C. (ice-bath) with 3-sulfosuccinimidyl4-maleimidobutyrate (1) as described above, to providemaleimido-derivatized protein containing an average of 29 maleimidogroups per molecule of protein.

[0092] Coupling Reaction:

[0093] The diene 27 was treated with the maleimido-derivatized proteinin water at 23° C. Samples taken at various times were diafilteredthrough a 10 kDa cutoff membrane then were lyophilized and subjected toMALDI-TOF mass spectrometry. The increase in molecular weight of theresulting conjugate 28 (n≦29), relative to the mass of the core proteinwith the dienophiles attached, is shown as a function of time in FIG. 1.

[0094] A similar time-dependent experiment was conducted with the samemaleimido-derivatized protein, using previously described diene 8, andthe results are shown in FIG. 2. Kinetic parameters are shown in Table2. TABLE 2 Kinetic Parameters for Conjugation Reactions Parameters forΔmw = Δmw_(max)(1 − e^(-kt)) Diene Δmw_(max) k (min⁻¹) t_(1/2) (min) 149000 0.007 99 8 4350 0.0045 154 EXAMPLE 6

[0095] Diene Component:

[0096] The previously described ester 16 was converted to hydrazide 17by treatment with hydrazine in MeOH at 23° C. for 24 hr.:¹H NMR (500MHz, CD₃OD): δ=6.00 (m, 2H), 5.57 (m, 1H), 5.49 (m, 1H), 2.33 (m, 2H),2.21 (m, 2H), 1.70 (d, 3H).

[0097] Acylation with methyl malonyl chloride in pyridine at −20° C. for20 min afforded the ester 29 in 72% yield, ¹H NMR (500 MHz, CD₃OD):δ=6.03 (m, 2H), 5.58 (m, 2H), 3.74 (s, 3H), 3.41 (m, 2H), 2.31-2.43 (m,4H), 1.23 (d, 3H).

[0098] Hydrolysis with LiOH in methanol (23° C., 1 hr), followed byacidification with 1 N HCl, gave the crystalline acid 30 in 74% yield,¹H NMR (500 MHz, CD₃OD): δ=5.97 (m, 2H), 5.52 (m, 2H), 3.26 (m, 2H),3.24-3.37 (m, 4H), 1.66 (d, 3H).

[0099] Treatment of the dodecasaccharide hydrazide 31 (V. Pozsgay, J.Org. Chem. 1998, 63:5983) with the linker 30 in DMF in the presence ofHATU led to the diene-equipped construct 32, which was purified bygel-filtration through a Biogel P-4 column using water as the eluant. ¹HNMR (500 MHz, D₂O): δ=6.11 (m, 2H), 5.72 (m, 1H), 5.61 (m, 1H), 5.60 (brs, 3H), 5.11 (br s, 2H), 5.08 (br s, 1H), 5.06 (br s, 2H), 5.04 (d, 2H),5.00 (d, 1H), 4.81 (br s, 1H), 1.71 (d, 3H); FAB-MS(dithiothreitol-dithioerythritol, positive ion) 2363.9 (M+Na), calcd.:2362.9.

[0100] Dienophile Component:

[0101] A stirred solution of human serum albumin in pH 7.5 buffer (1 ml)was treated at 5° C. (ice-bath) with 3-sulfosuccinimidyl4-maleimidobutyrate (1) as described above, to providemaleimido-derivatized protein containing an average of 22 maleimidogroups per molecule of protein.

[0102] Coupling Reaction:

[0103] Under conditions similar to those used for the conjugation ofconstructs 8 and 27, cycloaddition of 32 onto the maleimido-derivatizedprotein took place at a slower rate. After 2 hours, the averageincorporation was 1.5 dodecasaccharide chains per HSA molecule, andafter 8 hours, the incorporation reached an average of 3.

I claim:
 1. A method of coupling a first biomolecule to a secondbiomolecule, comprising: (a) covalently attaching a diene moiety to thefirst biomolecule to form a diene component; (b) covalently attaching adienophile to the second biomolecule to form a dienophile component; and(c) contacting the diene component with the dienophile component underconditions that permit a cycloaddition reaction to occur between thecomponents.
 2. A method of coupling a biomolecule to a gel or solidsupport, comprising: (a) covalently attaching a diene moiety to asubstrate selected from the group consisting of the biomolecule and thesupport, to form a diene component; (b) covalently attaching adienophile to the substrate not selected in step (a) to form adienophile component; and (c) contacting the diene component with thedienophile component under conditions that permit a cycloadditionreaction to occur between the components.
 3. The method of claim 1,wherein the first biomolecule is a polysaccharide and the secondbiomolecule is a polypeptide.
 4. The method of claim 3 wherein thepolysaccharide is selected from the group consisting of bacterialcapsular polysaccharides, fragments thereof, and synthetic analoguesthereof.
 5. The method of claim 4, wherein the bacterial capsularpolysaccharide is selected from the group consisting of capsularpolysaccharides of Haemophilus influenzae type b, Neisseriameningitidis, Group B Streptococci, Salmonella typhi, E. coli, andPneumococci.
 6. The method of claim 3 wherein the polypeptide isselected from the group consisting of bacterial toxins, bacterialtoxoids, bacterial outer membrane proteins, keyhole limpet hemocyanin,horseshoe crab hemocyanin, edestin, mammalian serum albumins, mammaliangamma-globulins, and IgG-G.
 7. The method of any one of claims 1-6wherein the dienophile moiety is attached to the biomolecule bycontacting the biomolecule with 3-sulfosuccinimidyl 4-maleimidobutyrate.8. The method of any one of claims 3-6 wherein the diene moiety isattached to the polysaccharide by glycosylation of trans,trans-hexa-2,4-dien-1-ol with the polysaccharide.
 9. The method of claim7 wherein one of the biomolecules is a polysaccharide, and the dienemoiety is attached to the polysaccharide by glycosylation of trans,trans-hexa-2,4-dien-1-ol with the polysaccharide.
 10. A conjugate ofbiomolecules prepared by the method of claim
 1. 11. A conjugate of abiomolecule with a solid or gel support, prepared by the method of claim2.
 12. A conjugate of biomolecules prepared by the method of any one ofclaims 3-6.
 13. A conjugate of biomolecules prepared by the method ofclaim
 7. 14. A conjugate of biomolecules prepared by the method of claim8.
 15. A conjugate of biomolecules prepared by the method of claim 9.16. A conjugate of biomolecules selected from the group consisting of

wherein R and R′ are independently H or methyl, or together constituteCH₂, CH₂CH₂, or O; X is CH or N; Y is N, CH═C, or NH—N; and B₁ and B₂comprise biomolecules independently selected from the group consistingof polypeptides, carbohydrates, polysaccharides, and nucleic acids, andare optionally attached via a linker.
 17. The conjugate of biomoleculesaccording to claim 16, wherein one of the biomolecules is apolysaccharide.
 18. The conjugate of biomolecules according to claim 17,wherein the polysaccharide is a viral or bacterial polysaccharide. 19.The conjugate of biomolecules according to claim 16, wherein one of thebiomolecules is a polysaccharide and the other biomolecule is apolypeptide.
 20. The conjugate of biomolecules according to claim 19,wherein the polysaccharide is a viral or bacterial polysaccharide. 21.An immobilized biomolecule selected from the group consisting of

wherein R and R′ are independently H or methyl, or together constituteCH₂, CH₂CH₂, or O; X is CH or N; Y is N, CH═C, or NH—N; one of B₁ and B₂comprises a biomolecule selected from the group consisting ofpolypeptides, carbohydrates, polysaccharides, and nucleic acids and theother of B₁ and B₂ is a solid or gel support, and B₁ and B₂ areoptionally attached via a linker.
 22. A pharmaceutical compositioncomprising a conjugate according to any one of claims 10, 11, or 16-20,further comprising a pharmaceutically acceptable carrier.
 23. Apharmaceutical composition comprising a conjugate according to claim 12,further comprising a pharmaceutically acceptable carrier.
 24. Apharmaceutical composition comprising a conjugate according to claim 13,further comprising a pharmaceutically acceptable carrier.
 25. Apharmaceutical composition comprising a conjugate according to claim 14,further comprising a pharmaceutically acceptable carrier.
 26. Apharmaceutical composition comprising a conjugate according to claim 15,further comprising a pharmaceutically acceptable carrier.
 27. A methodof inducing, in a mammal, antibodies which immunoreact with apolysaccharide, comprising administering to said mammal a compositionaccording to claim 22, wherein one of the biomolecules is apolysaccharide.
 28. A method of inducing, in a mammal, antibodies whichimmunoreact with a polysaccharide, comprising administering to saidmammal a composition according to claim
 23. 29. A method of inducing, ina mammal, antibodies which immunoreact with a polysaccharide, comprisingadministering to said mammal a composition according to claim 24,wherein one of the biomolecules is a polysaccharide.
 30. A method ofinducing, in a mammal, antibodies which immunoreact with apolysaccharide, comprising administering to said mammal a compositionaccording to claim
 25. 31. A method of inducing, in a mammal, antibodieswhich immunoreact with a polysaccharide, comprising administering tosaid mammal a composition according to claim
 26. 32. An antibody whichimmunoreacts with a polysaccharide, wherein said antibody is obtainedfrom a mammal, and wherein the production of the antibody by the mammalhas been induced by the method of claim
 27. 33. An antibody whichimmunoreacts with a polysaccharide, wherein said antibody is obtainedfrom a mammal, and wherein the production of the antibody by the mammalhas been induced by the method of claim
 28. 34. An antibody whichimmunoreacts with a polysaccharide, wherein said antibody is obtainedfrom a mammal, and wherein the production of the antibody by the mammalhas been induced by the method of claim
 29. 35. An antibody whichimmunoreacts with a polysaccharide, wherein said antibody is obtainedfrom a mammal, and wherein the production of the antibody by the mammalhas been induced by the method of claim
 30. 36. An antibody whichimmunoreacts with a polysaccharide, wherein said antibody is obtainedfrom a mammal, and wherein the production of the antibody by the mammalhas been induced by the method of claim
 31. 37. An antibody, produced bya hybridoma, which immunoreacts with a polysaccharide, wherein nucleicacid sequences encoding said antibody in said hybridoma are obtainedfrom a mammal in which the production of the antibody has been inducedby the method of claim
 27. 38. An antibody, produced by a hybridoma,which immunoreacts with a polysaccharide, wherein nucleic acid sequencesencoding said antibody in said hybridoma are obtained from a mammal inwhich the production of the antibody has been induced by the method ofclaim
 28. 39. An antibody, produced by a hybridoma, which immunoreactswith a polysaccharide, wherein nucleic acid sequences encoding saidantibody in said hybridoma are obtained from a mammal in which theproduction of the antibody has been induced by the method of claim 29.40. An antibody, produced by a hybridoma, which immunoreacts with apolysaccharide, wherein nucleic acid sequences encoding said antibody insaid hybridoma are obtained from a mammal in which the production of theantibody has been induced by the method of claim
 30. 41. An antibody,produced by a hybridoma, which immunoreacts with a polysaccharide,wherein nucleic acid sequences encoding said antibody in said hybridomaare obtained from a mammal in which the production of the antibody hasbeen induced by the method of claim
 31. 42. A method of inducing passiveimmunity in a mammal, comprising administering to said mammal aneffective amount of an antibody according to claim
 32. 43. A method ofinducing passive immunity in a mammal, comprising administering to saidmammal an effective amount of an antibody according to claim
 37. 44. Avaccine composition comprising a conjugate according to claim 12,further comprising an adjuvant and a pharmaceutically acceptablecarrier.
 45. A vaccine composition comprising a conjugate according toclaim 13, further comprising an adjuvant and a pharmaceuticallyacceptable carrier.
 46. A vaccine composition comprising a conjugateaccording to claim 14, further comprising an adjuvant and apharmaceutically acceptable carrier.
 47. A vaccine compositioncomprising a conjugate according to claim 15, further comprising anadjuvant and a pharmaceutically acceptable carrier.